The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided by the terms of cooperative agreement No. 70NANB7H3071 awarded by NIST.
In the present era of arterial replacement, at least 345,000-485,000 autologous coronary grafts (either arteries or veins) and over 200,000 autogenous vein grafts into peripheral arteries are performed each year. Report of a working party of the British Cardiac Society: Coronary Angioplasty in the United Kingdom. Br Heart J. 66:325-331, 1991; Heart and Stroke Facts: Statistical Supplement, American Heart Association, 1996; and Callow A. D. "Historical overview of experimental and clinical development of vascular grafts," In: Biologic and Synthetic Vascular Prosthesis, Stanley J (Ed), Grune and Stratton, New York, 11, 1983. A recent marketing report indicated that at least 300,000 coronary artery bypass procedures are performed annually in the United States involving in excess of 1 million vascular grafts. World Cell Therapy Markets, Frost & Sullivan, 5413-43 Revision #1, ISBN 0-7889-0693-3, 1997.
Many of these patients do not have autologous veins suitable for grafts due to pre-existing vascular disease, vein stripping or use in prior vascular procedures. It has been estimated that as many as 30% of the patients who require arterial bypass procedures will have saphenous veins unsuitable for use in vascular reconstruction. Edwards W. S., Holdefer W. F., Motashemi, M., "The importance of proper caliber of lumen in femoral popliteal artery reconstruction," Surg Gynecol Obstet. 122:37, 1966. More recently it has been demonstrated that 2-5% of saphenous veins considered for bypass procedures were unusable on the basis of gross pathology and that up to 12% were subsequently classified as diseased. These "diseased" veins had patency rates less than half that of non-diseased veins. Panetta T. F., Marin M. L., Veith F. J., et al., "Unsuspected pre-existing saphenous vein disease: an unrecognized cause of vein bypass failure," J Vasc Surg. 15:102-112, 1992. However, we estimate that if all arterial grafts and alternative veins are utilized according to current surgical practice, the maximum number of potential allograft recipients is probably closer to 10%.
Vitrified arterial grafts may also have a market as a scaffold for the seeding and adhesion of autologous endothelial cells or genetically modified endothelial cells. Prosthetic grafts are currently employed for large diameter (greater than 6mm internal diameter) non-coronary applications. Between 1985 and 1990, approximately 1,200 allogeneic vein segments were employed for arterial bypass. Brockbank K. G. M., McNally R. T., Walsh K. A., "Cryopreserved vein transplantation," J Cardiac Surg. 7:170-176, 1992. The demand for allogeneic veins is growing despite the well documented immune response to these grafts and the low clinical patency rates. In 1991 alone, at least 1,400 allograft saphenous vein segments were transplanted. McNally R. T., Walsh K., Richardson W., "Early clinical evaluation of cryopreserved allograft vein," Proceedings of the 29th meeting of the Society for Cryobiology, Cryobio., Abstract #4, 1992. Conservatively, the market potential for vitrified vascular grafts may be 50,000 units per year, or 10% of all vascular grafting procedures in the United States.
Blood vessels are also a ubiquitous component of vascularized tissues and organs, both human and animal, which may one day be successfully stored by vitrification for transplantation. Providing that significant immunological issues can be overcome, animal-derived grafts may, one day, provide an unlimited supply of blood vessels and vascularized tissues and organs that could be stored in a vitrified state prior to transplantation.
Low temperature preservation of biological tissues and organs, i.e., cryopreservation, has been the subject of much research effort. Cryopreservation can be approached by freezing or by vitrification. If the organ or tissue is frozen, ice crystals may form within the organ or tissue that may mechanically disrupt its structure and thus damage its ability to function correctly when it is transplanted into a recipient. Organized tissues and organs are particularly susceptible to mechanical damage from ice crystals formed during freezing.
Vitrification, by contrast, means solidification, as in a glass, without ice crystal formation. The principles of vitrification are well-known. Generally, the lowest temperature a solution can possibly supercool to without freezing is the homogeneous nucleation temperature T.sub.h, at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution. Vitrification solutions have a glass transition temperature T.sub.g, at which temperature the solute vitrifies, or becomes a non-crystalline solid. Owing to the kinetics of nucleation and crystal growth, it is effectively impossible for water molecules to align for crystal formation at temperatures much below T.sub.g. In addition, on cooling most dilute aqueous solutions to the glass transition temperature, T.sub.h is encountered before T.sub.g, and ice nucleation occurs, which makes it impossible to vitrify the solution. In order to make such solutions useful in the preservation of biological materials by vitrification, it is therefore necessary to change the properties of the solution so that vitrification occurs instead of ice crystal nucleation and growth. It is also important that all viability and tissue function be maintained by the entire vitrification process.
While it is generally known that high hydrostatic pressures raise T.sub.g and lower T.sub.h, vitrification of most dilute solutions by the application of pressure is often impossible or impractical. In particular, for many solutions vitrifiable by the application of pressure, the required pressures cause unacceptably severe injury to unprotected biomaterials during vitrification thereof While it is also known that many solutes, such as commonly employed cryoprotectants like dimethyl sulfoxide (DMSO), raise T.sub.g and lower T.sub.h, solution concentrations of DMSO or similar solutes high enough to permit vitrification typically approach the eutectic concentration and are generally toxic to biological materials.
One type of damage caused by cryoprotectants is osmotic damage. Cryobiologists learned of the osmotic effects of cryoprotectants in the 1950's and of the necessity of controlling these effects so as to prevent damage during the addition and removal of cryoprotectants to isolated cells and tissues. Similar lessons were learned when cryobiologists moved on to studies of whole organ perfusion with cryoprotectants. Attention to the principles of osmosis were essential to induce tolerance to cryoprotectant addition to organs.
Despite efforts to control the deleterious osmotic effects of cryoprotectants, limits of tolerance to cryoprotectants are still observed. There appear to be genuine, inherent toxic effects of cryoprotectants that are independent of the transient osmotic effects of these chemical agents.
Studies by the present inventors and others have examined methods of controlling non-osmotic, inherent toxicity of cryoprotectant agents. The results indicate that several techniques can be effective alone and in combination. These include (a) the use of specific combinations of cryoprotectant whose effects cancel out each other's toxicities; (b) exposure to cryoprotectants in vehicle solutions that are optimized for those particular cryoprotectants; (c) the use of non-penetrating agents that can substitute for a portion of the penetrating agent otherwise needed, thus sparing the cellular interior from exposure to additional intracellular agents; and (d) minimizing the time spent within the concentration range of rapid time-dependent toxicity.
Some of these techniques are in potential conflict with need to control osmotic forces. For example, reduced temperatures also reduce the influx and efflux rate of cryoprotectants, thereby prolonging and intensifying their osmotic effects. Similarly, minimizing exposure time to cryoprotectants maximizes their potential osmotic effects. Thus, there must be a balance reached between the control of osmotic damage and the control of toxicity. Means for obtaining this balance are described in U.S. Pat. No. 5,723,282 to Fahy et al. However, this patent does not describe a particular method to be used for blood vessels. In addition, this patent does not discuss any protocols for cooling or warming the organ or tissue.